Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, and 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 days. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (IGF-1R, VEGF, FGF2, and FGF7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter.